The present invention pertains generally to a process for purifying an enzyme and, more specifically, an improved process for obtaining a purified enzyme solution from a crude enzyme containing solution such as a cell extract liquor, a fermentation culture liquid or the like.
Heretofore, various processes have been employed for purifying enzymes, and processes using synthetic adsorbents, weakly basic anion exchange resins, weakly acidic cation exchange resins, and adsorbents such as cellulose, Sephadex, carbon, etc. are known. However, these processes suffer from various drawbacks which render them unsuitable for enzyme purification on a commercial scale. That is, in many processes using a synthetic adsorbent such as resins of the Amberlite XAD series (trademark for products of Rohm and Haas Co.), resins of the Diaion HP series (trademark for products of Mitsubishi Kasei Kogyo Co.), etc., the enzyme is either poorly adsorbed onto the resins by contact of an enzyme solution with the resins or, if adsorbed, is hardly eluted by an eluting agent. Moreover, the species of enzyme to which these processes are applicable are limited to protease, amylase, etc.
The processes using Sephadex having ion exchange groups are excellent in chromatographic separation, but a high flow rate cannot be obtained in column operation, and, thus, are not applicable to a commercial scale process. Ion exchange resins in the nature of weakly acidic cation exchange resin such as Amberlite IRC-50 and CG-50 (trademarks for products of Rohm and Haas Co.), weakly basic anion exchange resin such as Duolite A-7 (trademark for product of Diamond Shamrock Co.), etc. are known. However, since these ion exchange resins do not have the ability to decompose neutral salts and have a small specific surface area, they also have serious drawbacks. For example, the enzyme cannot be adsorbed onto the ion exchange resins or only a very small amount of enzyme can be adsorbed from a crude enzyme solution containing a large amount of impurities, or from a solution wherein the enzyme is dissolved in a solution of salts of high ionic intensity, a buffer solution, etc. Thus, these ion exchange resins are also not practical.
It has been proposed that strongly acidic cation exchange resins of macroreticular structure type such as AGMP-50 (trademark for a product of Bio . Rad Laboratory Co.) are applicable for adsorption and elution of an enzyme, but the adsorption rate is not specified and it is not certain that these resins are practically applicable.
On the other hand, it is known to use strongly basic anion exchange resins such as Amberlite IRA-904 and IRA-938 (trademarks for products of Rohm and Haas Co.) of macroreticular structure type in adsorption and elution of pepsin and analysis of creatinine kinase.
Therefore, a need exists for an improved process for purification of a crude enzyme solution which is applicable to a commercial scale. To this end, it has now been found that when various enzyme solutions are brought into contact with a strongly acidic cation exchange resin of high porous type or a strongly basic anion exchange resin of high porous type, the enzyme is adsorbed onto the ion exchange resin at a high adsorption rate, i.e. in a high adsorption amount, and when the resin is eluted with an appropriate eluting agent, the enzyme can be desorbed into the eluate in a high yield with a considerable increase in specific enzyme activity.